Merodrimanes and Other Constituents from Talaromyces thailandiasis

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Merodrimanes and Other Constituents from Talaromyces thailandiasis Tida Dethoup,†,‡ Leka Manoch,† Anake Kijjoa,‡ Madalena Pinto,§ Luis Gales,⊥ Ana Margarida Damas,⊥ Artur M. S. Silva,# Graham Eaton,3 and Werner Herz*,x Department of Plant Pathology, Faculty of Agriculture, Kasetsart UniVersity, Bangkok, Thailand, ICBAS-Instituto de Cieˆ ncias Biome´ dicas Abel Salazar and CIIMAR, UniVersidade do Porto, 4099-003, Porto, Portugal, Centro de Estudo de Quı´mica Orgaˆ nica, Fitoquı´micas e Farmacologia de UniVersidade do Porto (CEQOFFUP), Faculdade de Farma´ cia, Rua Anibal Cunha 164, 4050-047 Porto, Portugal, ICBAS-Instituto de Cieˆ ncias Biome´ dicas Abel Salazar and Unidade de Estrutura Molecular-IBMC, UniVersidade do Porto, 4094-003 Porto, Portugal, Departamento de Quı´mica, UniVersidade de AVeiro, 4810-1933 AVeiro, Portugal, Department of Chemistry, Leicester UniVersity, UniVersity Road, Leicester LE 7RH, U.K., and Department of Chemistry and Biochemistry, The Florida State UniVersity, Tallahassee, Florida 32306-4390 ReceiVed October 20, 2006

Chemical study of a previously undescribed fungus, Talaromyces thailandiasis, furnished the two new merodrimanes thailandolides A (1) and B (2), an O-methylated derivative (3) of the aromatic fragment incorporated in thailandolide B, and three known closely related 1(3H)-isobenzofuran derivatives, penisimplicissin (4a), vermistatin (4b), and hydroxydihydrovermistatin (4c). Structures were established by spectroscopic measurements and confirmed by X-ray analyses of compounds 1 and 4b. The unusual peptide analogue N-benzoylphenylalanyl-N-benzoylphenyl alaninate (5) isolated earlier from a higher plant was also found. Since the spiroketals talaromycins A-F from the ascomycetous fungus Talaromyces stipitatus were described more than 20 years ago,1-3 a spate of articles dealing with secondary metabolites from other members of the genus have appeared, the most recent one describing the isolation of wortmannilactones A-D from Talaromyces wortmannii.4 Almost all secondary metabolites isolated from Talaramyces species so far appear to be of polyketide origin, the exception being wortmannin, also from T. wortmannii,5 which appears to be a partially oxidized steroid. We have recently described two new oxyphenalenone dimers, bacillisporins D and E, from Talaromyces bacillisporus in addition to the previously known analogues duclauxin and bacillisporins A-C.6 We now report the isolation of two new merodrimanes (1 and 2) from a previously undescribed fungus, Talaromyces thailandiasis Manoch and Dethoup,7 as the first terpenoids found in this genus of Penicillium teleomorphs. Other metabolites produced by this fungus were the methyl ether 3 of the ketide half of 1, three closely related known γ-pyrones, 4a-c, incorporating a 1(3H)isobenzofuranone, N-benzoylphenylalanyl-N-benzoylphenylalaninate (5) previously reported by one of us from Croton hieronymi,8 and 2-glycerylpalmitate. That compound 1, which we have named thailandolide A, was a 3-oxo-7β-hydroxydrimane linked through a tertiary oxygen to an aromatic moiety incorporating a lactone function in the manner characteristic of merodrimanes known from fungi of the genus Stachybotrys,9 but with the lactone function closed to C-8 of the drimane portion as in the kampanols from Stachybotrys kampalensis,10 was deduced from the 1H and 13C NMR spectra, listed in Table 1. The probable stereochemistries of C-8 and C-8′ and the location of the phenolic hydroxyl group on C-4′ of the aromatic ring deduced from chemical shifts and coupling constants in Table 1 as well as COSY and NOESY data were confirmed by an X-ray analysis. The ORTEP diagram of 1 (Figure 1) led to the relative configuration shown in formula 1. 1H and 13C NMR spectroscopic data of compound 2 (thailandolide B) demonstrated that it differed from 1 in being the * To whom correspondence should be addressed. Tel: +1-850-644-2774. Fax: 1-850-644-8281. E-mail: [email protected]. † Kasetsart University. ‡ ICBAS, Universidade do Porto. § CEQOFFUP, Universidade do Porto. ⊥ IBMC, Universidade do Porto. # Universidade de Aveiro. 3 Leicester University. x Florida State University. 10.1021/np0680578 CCC: $37.00

corresponding enone that carried an extra β-oriented acetoxy group on C-7′ of the aromatic ring. An additional compound present in

© 2007 American Chemical Society and American Society of Pharmacognosy Published on Web 05/31/2007

Notes

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Table 1. NMR Data of Compounds 1 and 2 in CDCl3 (1H 300 MHz, 13C 75 MHz) 1 position 1R 1β 2R 2β 3 4 5 6R 6β 7 8 9 10 11R 11β 12a 13a 14a 15a 1′ 2′ 3′ 4′ 5′ 6 7′R 7β 8′ 9′ 10 7-OH 4′-OH Ac a

δH (J in Hz) 2.01c 1.7c 2.49c 2.65td (11, 4.8) 1.95dd (14,3) 1.59 2.18ddd (14, 7.5, 3) 4.03t (7.5) 2.05dd (12,6.2) 2.5c 2.5c 1.37s 1.11s 1.12s 1.05s

6.35s 2.86dd (16.5, 3.5) 2.72dd (16.5, 12) 4.65ddq (12, 3.5, 6.3) 1.56d (6.3)

2 δC (DEPT)

δH

δC

32.1t

7.16d (10.3)

156.2d

33.6t

6.00d (10.3)

127.5d

207.3s 46.9s 43.6d 28.2t 73.0d 78.3s 40.8d 35.7s 19.5t 23.1q 20.0q 28.6q 22.8q 102.2s 139.1s 109.9s 162.3s 103.1d 158.6s 31.7t 74.7d 21.0q 170.0s

3.11brs 11.09s

2.18dd (14, 4.6) 2.2-2.3c 1.85ddd (14,14,2.4) 4.18dd (8.6, 2) 2.27dd (14, 5) 2.62dd (15, 14) 2.96dd (15, 5) 1.27s 1.11s 1.12s 1.36s

6.50s 6.17d (1.7) 4.73qd (6.6, 1.7) 1.49d (6.6) 11.06s 2.17sa

203.7s 44.6s 42.4d 26.5t 71.8d 79.7s 41.8d 38.4s 21.5t 21.4q 21.3q 27.6q 27.4q 102.2s 135.8s 112.3s 162.2s 106.3d 159.6s 64.1d 76.0d 16.4q 168.8s 20.7q 170.6s

Intensity three protons.

Figure 1. ORTEP view of compound 1.

Figure 2. ORTEP view of compound 4b.

the extract was 3, an O-methylated derivative of the aromatic fragment incorporated in 2. The ring conformation of 3 is such that the methyl group is quasiequatorial, as can be gleaned from the H-3/H-4a,b coupling constants and conforms to the stereochemistry of the C-8′-methyl group in 1. The location of the hydroxyl group on C-6 and the methoxy group on C-8 is shown by the presence of hydrogen bonding to the carbonyl, as evidenced by a broad -OH signal at δ 10.29, the COSY spectrum, which exhibited cross-peaks between H-5 and H-1′a,b and between H-3 and the OMe group, and the HMBC spectrum with cross-peaks between H-5 and CH2-1′, C-3, C-1, and C-0, between H-3 and C-1, C-5, and CO, and between -OMe and CO. Three further constituents of the extract were three 1(3H)isobenzofuranone derivatives, 4a-c. Compound 4b was identical with vermistatin, which has been isolated previously from cultures of Penicillium Verticulatum,11,12 Verruculosum,13 and simplicissimum14 as well as from fungal cultures related to Talaromyces flaVus,15-17 while compounds 4a and 4c were identical with penisimplicissin and a hydrated analogue of vermistatin, both recently reported from Pencillium simplicissimum.14 The structure of 4b was confirmed by an X-ray analysis (Figure 2).

Experimental Section General Experimental Procedures. 1H and 13C NMR spectra were recorded at ambient temperature in CDCl3 on a Bruker AMC instrument operating at 300.13 and 75.47 MHz, respectively, or on a Bruker DRX instrument operating at 500 and 125 MHz, respectively. EI mass spectra were measured on a Hitachi Perkin-Elmer RMV-GM instrument. HR mass spectra were measured on a Kratos Concept II 2 sector mass spectrometer. The accelerating voltage was 8 kV. Melting points were recorded on a Bock monoscope and are uncorrected. Optical rotations were determined on a Polax-2L instrument. Si gel for chromatography was silica gel 60 (0.2-0.5 mm Merck) for analytical work and for preparative TLC Merck silica gel 60 GF 254. Fungal Material. T. thailandiasis was isolated by L.M. from a soil sample collected in Trat Province, Southern Thailand, in August 2003. The strain, accession number KPFC 3399, is on deposit in the Department of Plant Pathology, Faculty of Agriculture, Kasetsart University. Fermentation, Extraction, and Isolation. Fifty 1 L Erlenmeyer flasks, each containing 200 g of rice and 100 mL of H2O, were inoculated with 10 mycelium plugs from the T. thailandiasis culture and incubated at 28 °C for 30 days. At the end of this period and after the subsequent addition of 400 mL of ethyl acetate to each flask the

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mixture was allowed to stand for 3 days and filtered using filter paper. Concentration of the filtrate at reduced pressure to a volume of 3 L, addition of anhydrous sodium sulfate, filtration, and evaporation at reduced pressure furnished 79.2 g of dark brown viscous ethyl acetate extract, which was extracted with CHCl3 (3 × 500 mL). Combination of the CHCl3 extracts followed by evaporation at reduced pressure afforded 51.4 g of viscous CHCl3 extract. A portion (40 g) was applied to a Si gel column (200 g) and eluted with CHCl3-petroleum ether and CHCl3-acetone mixtures, 300 mL fractions being eluted as follows: Fractions 1-202 (CHCl3-petroleum ether, 1:1), 203-265 (CHCl3-petroleum ether, 7:3), 266-285 (CHCl3-petroleum ether, 9:1), 286-316 (CHCl3-acetone, 9:1), 317-343 (CHCl3-acetone, 4:1), and 344-368 (CHCl3-acetone, 7:3). Fractions 65-75 (345 mg) were combined and recrystallized from a mixture of CHCl3-petroleum ether to give 200 mg of 5 as colorless crystals: mp 214-215°, lit. mp 212.5-2138 [R]D24 +263 (CHCl3, c 0.38 g/100 mL); FABMS m/z 507 (M + H)+; 1H and 13C NMR spectra identical with those reported earlier.8 Fractions 77-90 and 93-95 were combined (179 mg). Recrystallization from CHCl3-petroleum ether afforded 4b (vermistatin, 59 mg), identified by HRMS, 1H and 13C NMR,14 and an X-ray crystallographic analysis (Figure 2). Combination of fractions 91 and 92 followed by TLC (Si gel, CHCl3-MeOH-HCO2H, 95:5:1) furnished 11.3 mg of 2 and an additional 20 mg of 4b. Combination of fractions 97-107 (244 mg) followed by recrystallization from CHCl3-petroleum ether afforded 1 (200 mg). Combination of fractions 111-130 (570 mg) and recrystallization from CHCl3-petroleum ether gave a yellow solid (45 mg), which was purified by TLC (Si gel, CHCl3-Me2O-HCO2H, 95: 5:1) to give 15.4 mg of 4a (penisimplicissin, 15.4 mg) identified by MS and 1H and 13C NMR spectrometry.14 Fractions 145-159 (121 mg) were combined and purified by TLC (Si gel, CHCl3-Me2O-EtOAcHCO2H, 85:10:5:1) to give 11.7 mg of 2-glycerylpalmitate identified by HRMS and 1H and 13C NMR spectrometry. Fractions 221-280 of the initial chromatogram were combined and chromatographed over Si gel (10 g) using CHCl3-petroleum ether, 100 mL fractions being collected as follows: fractions 1-50 (CHCl3petroleum ether, 1:1), 51-74 (CHCl3-petroleum ether, 7:3), 75-80 (CHCl3-petroleum ether, 9:1). Fractions 28-32 (127 mg) were combined and purified by TLC (Si gel, CHCl3-Me2O-HCO2H, 4:1: 0.1) to give 37 mg of 3. Fractions 286-313 (2.2 g) were combined and rechromatographed over Si gel (12 g), using CHCl3-petroleum ether mixtures and 100 mL subfractions as follows: subfractions 1-25 (CHCl3-petroleum ether, 7:3) and subfractions 26-50 (CHCl3petroleum ether, 9:1). Subfractions 16-24 were combined (220 mg) and purified by TLC (Si gel, CHCl3-Me2O-HCO2H, 4:1:0.1) to give 46 mg of 4c identified by MS and 1H and 13C NMR spectrometry.14 Thailandolide A (1): white, crystalline solid; mp 277-278 °C; [R]D24 -256 (CHCl3, c 0.039 g/100 mL); 1H and 13C NMR, see Table 1; + FABHRMS m/z 429.22778 (M + H)+ (calcd for C25H33O6, 429.22771); 1H and 13C NMR spectra in Table 1. Thailandolide B (2): white, crystalline solid; mp 274-275 °C; [R]D24 +134 (CHCl3, c 0.14 g/100 mL); 1H and 13C NMR, see Table 1; + FABHRMS m/z 485.21748 (M + H)+ (calcd for C27H33O8, 485.21754). 3-Methyl-6-hydroxy-8-methoxy-3,4-dihydroisocoumarin (3): gum; [R]D27 -46.5 (CHCl3, c 0.043 g/100 mL); 13C NMR (CDCl3) δ 162.9 (C, C-8), 162.8 (C, C-1), 161.3 (C, C-6), 144.0 (C, C-4a), 106.2 (CH, C-5), 104.6 (C, C-8a), 98.4 (CH, C-7), 72.9 (CH, C-3), 35.5 (CH2, C-4), 20.4 (CH3); 1H NMR 10.52 (brs, OH), 6.37 (s, H-7), 6.26 (s, H-5), 4.41 (m, H-3), 3.74 (s, 3p, OMe), 2.86 (dd, 16, 3.1, H-4) 2.27 (dd, 16, 10.9, H-4b), 1.32 (3p, d, 6.2, 3-Me), + FABHRMS m/z 209.08148 (M + H)+ (calcd for C11H13O4, 209.08138). X-ray Crystal Structure of 1. Suitable crystals were obtained by slow evaporation of a solution in chloroform-petroleum ether and were orthorhombic, space group P212121, cell volume V ) 2271.2(10) Å, a ) 11.0763(3) Å, b ) 12.831(3) Å, c ) 15.981(4) Å (uncertainties in parentheses). There were four molecules per unit cell, calculated density 1.253 g/cm3. Diffraction data were collected at 293 K with a Stoe IPDS plate equipped with Mo KR radiation (d ) 0.71073 Å; 3066 independent reflections were measured, of which 2057 were observed (I > 2σ(I)). The structure was solved by direct methods using SHELXS9718 and refined with SHELXL-97.19 Hydrogen atoms bonded to C-5, C-7, C-9, C-5′, and C-8′ were refined freely with isotropic radiation parameters. The rest of the hydrogen atoms were positioned with idealized geometry and refined attached to their parent C or D atoms. The refinement converged to R (I > 2σ(I)) ) 8.29% and wR2 (I >

Notes

2σ(I)) ) 23.25%. An ORTEP view is shown in Figure 1. Full details of the data collection, refinement, and tables of atomic coordinates, bond lengths and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre.20 X-ray Crystal Structure of 4b. Suitable crystals were obtained by slow evaporation of a solution in chloroform-petroleum ether and were triclinic, space group P1, cell volume V ) 769.1(4) Å, a ) 4.5194(12) Å, b ) 8.666(2) Å, c ) 19.755(6) Å, R ) 86.73(3)°, β ) 86,13(3)°, and γ ) 86.13(3)° (uncertainties in parentheses). There were two molecules per unit cell, calculated density 1.418 g/cm3. Diffraction data were collected as described in the previous paragraph. A total of 3450 independent reflections were measured, of which 2689 were observed (I > 2σ(I)). The structure was solved as described in the previous paragraph. Carbon and oxygen atoms were refined freely with isotropic displacement parameters. The rest of the hydrogen atoms were positioned with idealized geometry and refined attached to their parent C or D atoms. The refinement converged to R (I > 2σ(I)) ) 5.51% and wR2 (I > 2σ(I)) ) 14.62%. An ORTEP view is shown in Figure 1. Full details of the data collection and refinement and tables of atomic coordinates, bond lengths and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre.20 Acknowledgment. Work in Portugal was supported by Fundac¸ a˜o para a Cieˆncia e Tecnologia of Portugal (Unidade de I & D 226/96), POCT (QAIII), FEDER, and CIIMAR Pluriannual. T.D. thanks the Thailand Research Fund (TRF) for a fellowship under the RGJ-PhD program. References and Notes (1) Lynn, D. G.; Phillips, N. J.; Hutton, W. C.; Shabanowitz, J. J.; Fennell, D. I.; Cole, R. J. J. Am. Chem. Soc. 1982, 104, 7319-7322. (2) Hutton, W. C.; Phillips, N. J.; Graden, D. W.; Lynn, D. G. Chem. Commun. 1983, 864-866. (3) Phillips, N. J.; Cole, R. J.; Graden, D. W.; Lynn, D. G. Tetrahedron Lett. 1987, 28, 1619-1622. (4) Dong, Y.; Yang, J.; Zhong, H.; Lin, J.; Ren, X.; Liu, M.; Lu, X.; He, J. J. Nat. Prod. 2004, 69, 128-130. (5) Nakanishi, S.; Kakita, S.; Takahashi, I.; Kawahara, K.; Tsukuda, E.; Sano, T.; Yamada, R.; Yoshida, M.; Kase, H.; Matsuda, Y. J. Biol. Chem. 1992, 267, 2157-2163. (6) Dethoup, T.; Manoch, L.; Kijjoa, A.; Nascimento, M. S. J.; Puapairoj, P.; Silva, A. M. S.; Eaton, G.; Herz, W. Planta Med. 2006, 72, 957960. (7) A full description of this new species, which belongs to section Talaromyces series Flavi, will appear in the mycological literature. (8) Catala´n, C. A. N.; de Heluani, C. S.; Kotowicz, C.; Gedris, T. E.; Herz, W. Phytochemistry 2003, 64, 625-629. (9) Sawadjoon, S.; Kittakoop, P.; Isaka M.; Kirtikara, K.; Madla, S.; Thebtaranonth, Y. Planta Med. 2004, 70, 1085-1087, and references therein. (10) Singh, S. B.; Zink, D. L.; Williams, M.; Polishook, J. D.; Sanchez, M.; Silverman, K. C.; Lingham, R. B. Biorg. Med. Chem. Lett. 1998, 8, 2071-2076. (11) Fuska, J.; Uhrin, D.; Proksa, B.; Voticky, Z.; Ruppeldt, J. J. Antibiot. 1986, 39, 1605-1608. (12) Massias, M.; Molho, L.; Rebuffat, S.; Cesario, M.; Guilhen, J.; Pascard, C.; Bodo, B. Phytochemistry 1989, 28, 1491-1494. (13) Murtaza, N.; Husain, S.; Sarfaraz, T. B.; Sultana, N.; Faizi, S. Planta Med. 1997, 63, 191. (14) Komai, S.; Hosoe, T.; Itabashi, T.; Nozawa, K.; Yaguchi, T.; Fukushima, K.; Kawai, K. Heterocycles 2005, 65, 2771-2776. (15) Wang, H.; Atai, M.; Tomoda, H.; Omura, S. Zhongguo Kangshengsu Zazhi 2001, 26, 89-93; Chem. Abstr. 2002, 136, 247422. (16) Arai, M.; Tomoda, H.; Okuda, T.; Wang, H.; Tabata, N.; Masuma, R.; Yamaguchi, Y.; Omura, S. J. Antibiot. 2002, 55, 172-180; Chem. Abstr. 2002, 136, 337619. (17) Komai, S.; Hosoe, T.; Itabashi, T.; Nozawa, K.; Okada, K.; de Campos Takaki, G. M.; Chikamori, T.; Fukushima, K.; Miyaji, M.; Kawai, K. Mycotoxins 2004, 54, 15-19; Chem. Abstr. 206, 144, 28907. (18) Sheldrick, G. M. SHELXL-97, Program for the solution of crystal structures; University of Go¨ttingen: Germany, 1997. (19) Sheldrick, G. M. SHELXL-97, Program for the solution of crystal structures; University of Go¨ttingen: Germany, 1997. (20) Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge C82 IE2, UK (fax: +44-(0)223-336033 or e-mail: [email protected]). Deposition numbers are CCDC 641403 and 641484.

NP0680578